Cancer cell motility, chemotaxis as well as its ability to transmigrate through an endothelium layer play important roles in cancer cells'metastatic cascade. Cancer metastasis is a dynamic and complex process, it involves cancer cells leaving the primary tumor, entering blood circulation, arresting in blood vessel, invading a distant organ, and growing a new tumor. Rather than primary tumors, metastases are responsible for most cancer deaths. Despite their clinical importance, cancer metastases remain poorly understood. Current gene/protein expression profiling work has revealed many molecular factors that are responsible for cancer metastases. Intra-vital cell imaging in animal models has, for the first time, connected the cancer cell metastatic behavior directly with the molecular mechanism in vivo and provided insights into the cancer cell metastatic pathways. Despite of all the advances in our understanding of the cancer metastasis processes, inhibitors derived from these studies have either lacked specificity and/or been ineffective clinically. This is, in part, due to our lack of understanding that cancer cells never act alone. They are actively interacting with the microenvironment via the secretion of chemokines, growth factors, as well as the remodeling of the extracellular matrices (ECM). The understanding of the intricate interactions among different cell types and the extracellular matrices has becoming a critical component towards the eventual understanding of cancer metastases. We propose to bring together expertise on micro-chemical, micro-mechanical engineering and imaging (Dr. Wu), vascular vessel and cancer cell biology (Dr. Swartz) and cancer cell biology (Dr. Yen) to the challenges in both fundamental cancer cell biology and its potential applications in clinical chemotherapy for cancer metastases. Our short term goal is to build a physiologically relevant (3D), mechanically and chemically tunable in vitro model, and to bring the two important steps in cancer metastasis steps, migration and intravasation, under the light. Our long term goal is to find the key molecular players in the tumor cell microenvironment that underlie the cancer cell's metastatic behavior. To achieve this, we propose the following Specific Aims Aim1: To develop a 3D high throughput, hydrogel based, microfluidic in vitro model, with tunable micro- chemical and micro-mechanical environments, for mimicking two important steps in cancer cell metastasis - tumor cell migration within a ECM and intravasation from a 3D ECM through a vascular vessel. Aim2: To develop a computation algorithm, in conjunction with a newly developed 4D imaging technique, to automatically score the tumor cell invasiveness (characterized by cell motility, chemotaxis and cell transmigration rate). This Aim is critical in the realization of a truly high throughput system. Aim3: To assess quantitatively the tumor cell invasiveness in vitro under the influences of various chemokines, growth factors, ECM compositions, as well as the presence/absence of immune cells and stromal cells. PUBLIC HEALTH RELEVANCE: Metastases are responsible for most cancer deaths. The proposed work will use the emerging microfluidic technology in conjunction with a novel 4D alive cell tracking imaging technique to investigate roles of microenvironments in cancer cell invasiveness. Experimental results will advance the basic cancer cell biology;and it will also generate microfluidic in vitro tools that will find direct applications for high throughput cancer inhibitor screening.